Commensal production of a broad-spectrum and short-lived antimicrobial peptide polyene eliminates nasal Staphylococcus aureus

Antagonistic bacterial interactions often rely on antimicrobial bacteriocins, which attack only a narrow range of target bacteria. However, antimicrobials with broader activity may be advantageous. Here we identify an antimicrobial called epifadin, which is produced by nasal Staphylococcus epidermidis IVK83. It has an unprecedented architecture consisting of a non-ribosomally synthesized peptide, a polyketide component and a terminal modified amino acid moiety. Epifadin combines a wide antimicrobial target spectrum with a short life span of only a few hours. It is highly unstable under in vivo-like conditions, potentially as a means to limit collateral damage of bacterial mutualists. However, Staphylococcus aureus is eliminated by epifadin-producing S. epidermidis during co-cultivation in vitro and in vivo, indicating that epifadin-producing commensals could help prevent nasal S. aureus carriage. These insights into a microbiome-derived, previously unknown antimicrobial compound class suggest that limiting the half-life of an antimicrobial may help to balance its beneficial and detrimental activities.


Supplementary Information
Analysis of the epifadin BGC with antiSMASH 5.0 28 predicted a three-partite composition with an Nterminal NRP part followed by a PK moiety and a C-terminal single amino acid residue (Fig. 1c).
The NRPS1 (EfiA) enzyme was predicted to start the biosynthesis with an aromatic amino acid.Since the first adenylation domain (A-domain) is followed by two condensation domains (C-domain) the first amino acid was suggested to be incorporated twice, with the second to be converted to Dconfiguration by the second C-domain with its epimerization domain (Fig. 1c).The second A-domain of EfiA was predicted to incorporate aspartate, the third asparagine, which is predicted again to be epimerized to D-asparagine.The following amino acid position did not yield a clear prediction.
According to antiSMASH it could be a small or an aliphatic amino acid such as glycine, alanine, valine, leucine, isoleucine, or amino-butyric acid.
After stabilizing pure epifadin in DMSO-PA (palmitoyl ascorbate) solution, samples were applied immediately to antimicrobial activity assays.Nevertheless, chemical analysis by coupled HPLC-UV/VIS-MS of the intact compound revealed a continuous degradation of epifadin after prolonged incubation at room temperature within hours with the N-terminal peptide-amide as main degradation product.Storage at -80°C tremendously improved the stability of epifadin in DMSO-PA.
Tandem MS (MS/MS) experiments of intact epifadin yielded specific fragmentation patterns that supported the predicted peptide sequence (Extended Data Fig. 6).The determined structure of the peptide amide was further confirmed by acetylation and esterification reactions with the natural product extract (Extended Data Fig. 9).According to antiSMASH and the module organization of EfiA, the peptide amide should have an L-D-L-D-amino acid configuration (Fig. 1 and Extended Data Fig. 1).
L-Phe-D-Phe-L-Asp-D-Asn-NH 2 (FfDn-NH 2 ) and its enantiomer D-Phe-L-Phe-D-Asp-L-Asn-NH 2 (fFdN-NH 2 ) were synthesized by chemical solid-phase synthesis, employing the respective D-or L-asparagine rinkamide resin, and purified.The retention times on a HPLC-RP-C 18 column as well as the MS/MS spectra of the synthetic molecules were in accordance with the ones of the natural tetra peptide amide (Extended Data Fig. 1).In order to unambiguously confirm the absolute stereo configuration of the peptide moiety, the natural peptide amide was isolated from a decomposed epifadin NMR sample and both synthetic peptides and the natural peptide were analyzed by HPLC coupled MS using a column with a zwitterionic chiral stationary phase (CHIRALPAK® ZWIX(+)).By comparing the retention times the L-Phe-D-Phe-L-Asp-D-Asn-NH 2 was confirmed as the correct stereo configuration (Supplementary Fig. 2).Towards structure elucidation, a combination of 1 H-NMR and 2D-NMR experiments ( 1 H-1 H-ROESY, 1 H-1 H-COSY, and 1 H-13 C-HMBC) fully assigned and confirmed the presence of four unmodified amino acids, the N-terminal L-Phe, followed by D-Phe, L-Asp, and D-Asn, consistent with the sequence deduced from MS/MS (Extended Data Figs.3,4a).Additionally, NMR revealed the fifth amino acid to be a modified L-alanine (Extended Data Fig. 3).This adjacent Furthermore, correlations of 2D-NMR spectra support the tetramic acid structure (Supplementary Figures 3 and 4).Typical 13 C-NMR signals of the keto and enol groups of tetramic acid are in the range between 170 ppm and 200 ppm (see penicillenol G1 and G2 75 , more recently MCA17-1 76 ), which can also be found in 2D-NMR spectra of epifadin (1).However distinct signals of the tetramic acid moiety were not found.This phenomenon was also observed by another research group, which could not detect distinct signals in tetramic acid compounds such as militarinone C (3) 77 or pyranonigrin I (5) and J (4) 78 .
Typical derivatization approaches such as acetylation with acetic anhydride and pyridine or methylation with trimethylsilyldiazomethane in toluene/methanol in order to stabilize epifadin (1) has led to its decomposition.However, derivatized peptide amide fragment of 1 was observed via HPLC-HRMS analysis (Supplementary Figure 5).We also tested reutericyclin (6) and kirromycin (7) in a methylation reaction with trimethylsilyldiazomethane in toluene/methanol to optimize conditions for the methylation reaction of a purified epifadin (1) sample.Interestingly, only the methylation of the pyridine-ring containing kirromycin analogue yielded mono-as well as di-methylated kirromycin analogue (Supplementary Figure 7).The methylation of the tetramic acid reutericyclin (6) under the same conditions did not yield any mono-or multiple-methylated reutericyclin analogues (Supplementary Figure 6), which indicates that a tetramic acid might not be prone to this methylation method, which is the same in the case for methylation reaction of epifadin (1).
Since the derivatization reactions of epifadin (1) only led to its decomposition we used additives in the NMR solvents with the aim to stabilize one tautomer and prevent its decomposition.Additives such as ZnCl 2 , formic acid, or trifluoroacetic acid yielded only NMR spectra, which could not be analyzed.Changing the solvent to deuterated acetonitrile or methanol, we observed that epifadin (1) is poorly soluble in these solvents.Using formic acid or trifluoroacetic acid as additives resulted in quick dissolution of the residue.However, recording NMR spectra of the given solution resulted in decomposition.
EfiB represents a putative discrete AT/free-standing ACP S-malonyltransferase. AntiSMASH suggested that the PK extender units of the EfiB AT domain are derived from malonyl-CoA (Fig. 1c,d).This assumption is supported by the presence of the GHSxG motif (amino acid position 90-94 in EfiB), which is usually conserved in functional AT domains and also by the substrate-binding motif NAFH Since five individual S. epidermidis isolates with the nearly identical plasmid were isolated from three different geographical locations, frequent horizontal gene transfer (HGT) seems obvious.To identify a putative mechanism of HGT, plasmid sequences were further analyzed for the presence of genes involved in transfer.A putative relaxase gene of the newly described Firmicutes-specific MobL-family of relaxases was identified next to an origin of transfer (oriT) region, which clearly suggests horizontal dissemination by conjugation 81 .

Supplementary Figure 2 .
Extracted ion chromatograms (EICs, m/z = 541.2405± 0.05) of the analyses of the peptide amides were recorded with a HPLC-ESI-QTOF-MS.All pure samples had a concentration of 0.1 mg/mL.(a) The EIC of the synthetic L-D-L-D peptide (green).(b) The EIC (blue) shows a mixture (70/30) of the isolated natural peptide and the synthetic L-D-L-D peptide.(c) A mixture (70/30) of the isolated natural peptide and the synthetic D-L-D-L peptide is shown (EIC, red).(d) The EIC of the natural peptide purified from culture extract is shown (black).(e) The olive EIC shows a ternary mixture (30/15/50) of the synthetic L-D-L-D peptide, synthetic D-L-D-L peptide and the isolated natural peptide.(f) The mixture (30/70) of the synthetic L-D-L-D peptide and synthetic D-L-D-L peptide shows the given EIC (orange).(g) The EIC of the synthetic D-L-D-L peptide is presented (purple). of 2D-NMR experiments of epifadin (1) with focus on its tetramic acid.(A) In the structure of epifadin (1), arrows in blue show ROESY correlations, arrows in green show HMBC correlations, bold black bonds show COSY correlations and bold purple bonds show HSQC correlations. 1 H-(blue) and 13 C-NMR (red) shifts are given in ppm.(B) 1 H-1 H-COSY spectrum of epifadin (1) in DMSO-d 6 (700 MHz, 303 K). (C) 1 H-13 C-HMBC spectrum of epifadin (1) in DMSO-d 6 at 303 K (700 MHz).(D) Expansion of 1 H-13 C-HMBC spectrum of epifadin (1) in DMSO-d 6 at 303 K (700 MHz).Key correlation of tetramic acid moiety tautomer at 197.9 ppm ( 13 C) and 2.21 ppm ( 1 H).The tetramic acid moiety of epifadin (1) is assigned with its three carbonyl groups from the NMR-experiments in dmso-d 6 .1. Methylation reaction experiments towards methylated epifadin (1) with its tetramic acid moiety.Supplementary Figure 5. HPLC-HRMS analysis of the methylation reaction products of epifadin (1) with trimethylsilyldiazomethane.Chemical methylation of epifadin yielded only the methylated peptide and no methylated full epifadin analogue.Any polyene-tetramic acid fragments or analogues thereof were not detected.Base peak chromatogram of the reaction mixture is shown in grey, EIC of the peptide amide 2 is shown in blue and EIC of the methylated peptide amide 2 is shown in orange.Structures of the quasimolecular ion of the peptide amide 2 is shown in blue and of the methylated peptide amide 2 is shown in orange.TDB59_TMSCHN2_Epifadin_RB4_01_63673.d:BPC +All MS TDB59_TMSCHN2_Epifadin_RB4_01_63673.d: EIC C26H32N6O7 [M+H]+ 541.2405±0.02All MS TDB59_TMSCHN2_Epifadin_RB4_01_63673.d: EIC C27H34N6O7 [M+H]+ 555.2562±0.reactionexperiments towards methylated reutericyclin (6) with its tetramic acid moiety.Supplementary Figure 6.HPLC-HRMS analysis of the methylation reaction of reutericyclin (6) with trimethylsilyldiazomethane.EIC of reutericyclin (6) is shown in blue, EIC of the tetramic acid head (decomposition) is shown in orange, EIC of the amide chain of reutericyclin (impurity) is shown in green, EICs of mono methylated and dimethylated reutericyclin is shown in red, and UVchromatogram (280 nm) is shown in black.Left, reaction at t = 0 h.Right, reaction at t = 24 h.Two new unknown peaks can be observed in the UV-chromatogram at 15.3 min and 17.5 min, which cannot be assigned to any methylation or degradation product.Quasimolecular ions of the tetramic acid head (decomposition, orange) and the amide chain of reutericyclin (impurity, green) are shown.

3.
Methylation reaction to methylated pyridone bearing kirromycins.Supplementary Figure 7. HPLC-HRMS analysis of the methylation reaction of kirromycin (6) with trimethylsilyldiazomethane.EIC of kirromycin (7) is shown in blue, EIC of mono methylated kirromycin is shown in orange, EIC of di-methylated kirromycin is shown in green and UVchromatogram (350 nm) is shown in black.Left, reaction at t = 0 h.Center, reaction at t = 4 h.Right, reaction at t = 24 h after adding an additional 8 eq trimethylsilyldiazomethane.Supplementary Figure 8. Role of S. aureus desK genotype in S. epidermidis IVK83 growth inhibition.Central spots of S. epidermidis strains (IVK83, IVK83 efiTP and IVK83 efiTP pRB474-efiTP) were cultured simultaneously on agar plates spread with either S. aureus wild type or desK variant strains.
modified L-Ala residue lacks the C-terminal carbonyl group (C=O) as indicated by the NMR signals for the corresponding enamide moiety to resemble the -NH-C(CH 3 )=CH-structure. 1H-1 H correlation spectra (Extended Data Fig. 3 and 4) support a tetraene moiety attached to the peptide moiety and indicate an all-trans configuration.

(
amino acid position 197-200 in EfiB), specific for malonyl-CoA79 .The modules of EfiC and EfiD are probably responsible for the iterative condensation of the acetate extender units, which remain probably unsaturated because EfiC and EfiD contain ketosynthase (KS), peptidyl carrier protein (P), dehydratase (DH) and ketoreductase (KR) domains, of which the latter two generally catalyze double bond formation in polyketides.The PKS module of EfiF consists only of a KS and an ACP domain.The exact chemical structure of the PK part of epifadin could not be fully elucidated by only analytical methods as MS/MS or NMR spectroscopy, because of the extraordinary instability of epifadin.The naturally occurring and ionization-induced fragments are unsaturated PK moieties, which are difficult to detect by MS because they are often not prone to ionization.The unusual UV absorption maximum at 383 nm supports the presence of an unsaturated polyene PK moiety (Fig.3).Similar, albeit not fully identical UV absorption properties have also been found in the macrocyclic PK compound amphotericin B80 , which contains seven conjugated double bonds, in the antifungal PK sugar macrocyclic agent nystatin A1 with four plus two conjugated double bonds, and in militarinone C with four conjugated double bonds representing a polyenoyl tetramic acid77 .antiSMASHpredicted that the adenylation domain A 5 of the NRPS module of EfiE is specific for activating aspartate, which is subsequently linked to the PK part of epifadin.HPLC-coupled high resolution MS (MS/MS) analyses of epifadin indicated the modification of aspartate, to yield a tetramic acid as structural feature of epifadin.MS analyses delivered a characteristic signal pattern for the tetramic acid moiety, containing ions with well-assigned fragment ions (e.g.,[M+H] + , C 6 H 8 NO 4 + , calculated m/z 158.0448, found 158.0454, Δ3.9 ppm; [M+H] + , C 6 H 6 NO 3 + , calculated m/z 140.0342, found 140.0348, Δ4.1 ppm) (Extended DataFig.5).The proposed, chemical fragmentation mechanisms corresponded to the fragmentation observed by MS/MS, thereby supporting the heterocyclic tetramic acid structure (Extended Data Fig. 5).Unfortunately, the method of NMR gave only an incomplete set of signals, presumably as a result of pronounced tautomeric effects of the distinctly charged tetramic acid tautomers.Nevertheless, the genetic architecture of the BGC suggests that the release of the final molecule from the terminal NRP domain is probably catalyzed by cyclization to form the tetramic acid via the terminal C-domain of EfiE and, presumably, the thioesterase EfiT (Fig. 1).Condensation of the PK part with the aspartate residue and the subsequent Dieckmann cyclization catalyzed by the unusual C-terminal condensation domain of EfiE could form the tetramic acid moiety in analogy to a mechanism postulated for malonomycin biosynthesis 30 .